PROTEOMICS IN GRAM-NEGATIVE BACTERIAL OUTER
MEMBRANE VESICLES
Eun-Young Lee,1 Dong-Sic Choi,1 Kwang-Pyo Kim,2 and Yong Song Gho1*
1
Department of Life Science and Division of Molecular and Life Sciences,
Pohang University of Science and Technology, Pohang, Republic of Korea
2
Institute of Biomedical Science and Technology, Department of Molecular
Biotechnology, Konkuk University, Seoul, Republic of Korea
Received 12 December 2007; accepted 28 February 2008
Published online 17 April 2008 in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/mas.20175
Gram-negative bacteria constitutively secrete outer membrane such as nanotubes (Ratajczak et al., 2006). Recently, a
vesicles (OMVs) into the extracellular milieu. Recent research mechanism mediated by membrane vesicles (MVs), which are
in this area has revealed that OMVs may act as intercellular spherical, bilayered proteolipids with an average diameter of
communicasomes in polyspecies communities by enhancing 0.03 1 mm, has drawn much attention (Beveridge, 1999;
bacterial survival and pathogenesis in hosts. However, the Ratajczak et al., 2006). The secretion of MVs is a universal
mechanisms of vesicle formation and the pathophysiological cellular process occurring from simple organisms to complex
roles of OMVs have not been clearly defined. While it is multicellular organisms, including humans (Thery, Zitvogel,
obvious that mass spectrometry-based proteomics offers great & Amigorena, 2002; Mashburn-Warren & Whiteley, 2006).
opportunities for improving our knowledge of bacterial OMVs, Throughout evolution, both prokaryotic and eukaryotic cells
limited proteomic data are available for OMVs. The present have adapted to manipulate MVs for intercellular communi-
review aims to give an overview of the previous biochemical, cation via outer membrane vesicles (OMVs) in the case of
biological, and proteomic studies in the emerging field of Gram-negative bacteria and microvesicles in eukaryotic cells.
bacterial OMVs, and to give future directions for high- Increasing evidence suggests that MVs act as potent communi-
throughput and comparative proteomic studies of OMVs that casomes, that is, nano-sized extracellular organelles that play
originate from diverse Gram-negative bacteria under various diverse roles in intercellular communication (Choi et al., 2007),
environmental conditions. This article will hopefully stimulate and that the biogenesis and functions of MVs may share many
further efforts to construct a comprehensive proteome data- features in different biological systems. Thus, the study of MVs
base of bacterial OMVs that will help us not only to elucidate provides crucial keys to understanding the intercellular commu-
the biogenesis and functions of OMVs but also to develop nication network in living organisms and the evolutionary
diagnostic tools, vaccines, and antibiotics effective against connections between prokaryotes and eukaryotes (Mashburn &
pathogenic bacteria. # 2008 Wiley Periodicals, Inc., Mass Whiteley, 2005).
Spec Rev 27:535 555, 2008 A wide variety of Gram-negative bacteria constitutively
Keywords: outer membrane vesicles; gram-negative bacteria; secrete OMVs during growth (Beveridge, 1999), including
communicasomes; antibiotics; proteomics; vaccines Escherichia coli, Neisseria meningitidis, Pseudomonas aerugi-
nosa, Shigella flexneri, and Helicobacter pylori (Devoe &
Gilchrist, 1973; Hoekstra et al., 1976; Fiocca et al., 1999;
Kadurugamuwa & Beveridge, 1999). OMVs are spherical,
I. INTRODUCTION
bilayered proteolipids with an average diameter of 20 200 nm;
they are composed of outer membrane proteins, lipopolysac-
Communication between cells and the environment is an
charide (LPS), outer membrane lipids, periplasmic proteins,
essential process in living organisms, and intercellular commu-
cytoplasmic proteins, DNA, RNA, and other factors associated
nication is believed to be mediated mainly by the secretion of
with virulence (Horstman & Kuehn, 2000; Wai et al., 2003;
soluble factors, cell-to-cell contacts, and tunneling machinery
Kuehn & Kesty, 2005; Bauman & Kuehn, 2006; Nevot et al.,
2006; Lee et al., 2007). Studies of OMVs from diverse bacterial
strains suggest their roles in the delivery of toxins to host cells, the
   
Contract grant sponsor: National R&D Program for Cancer Control, transfer of proteins and genetic material between bacterial cells,
Ministry of Health & Welfare, Republic of Korea; Contract grant
cell-to-cell signals, and the elimination of competing organisms
number: 0320380-2; Contract grant sponsor: Korea Basic Science
(Kuehn & Kesty, 2005; Mashburn-Warren & Whiteley, 2006).
Institute K-MeP; Contract grant number: T27021; Contract grant
Because OMVs are essential to bacterial survival and patho-
sponsor: Korea Science and Engineering Foundation (KOSEF)
genesis in the host, modulation of vesicle formation and their
(MOST); Contract grant number: R15-2004-033-05001-0; Contract
functions may be a useful objective in relation to the development
grant sponsor: Brain Korea 21 fellowship.
of antibiotics (Henry et al., 2004; Lee et al., 2007).
*Correspondence to: Yong Song Gho, Department of Life Science,
Although recent research in this area has revealed the diverse
Division of Molecular and Life Sciences, Pohang University of Science
functions of OMVs, the mechanisms of vesicle formation and of
and Technology, San31 Hyojadong, Pohang, Kyungbuk 790-784,
protein sorting into OMVs, as well as the pathophysiological
Republic of Korea. E-mail: [e-mail]
Mass Spectrometry Reviews, 2008, 27, 535 555
# 2008 by Wiley Periodicals, Inc.
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LEE ET AL.
roles of OMVs, have not been clearly defined. To address these evolved several secretion pathways, including some mechanisms
issues, vesicular proteins should be comprehensively identified. common among human and plant pathogens (Lory, 1992).
Proteomics offers a powerful approach to decode the protein Approximately 20% of the polypeptides synthesized by bacteria
components of OMVs. Mass spectrometry (MS)-based proteo- are located partially or completely outside of the cytoplasm
mic studies have been used in human microvesicles to identify following secretion (Pugsley, 1993; Kostakioti et al., 2005).
thousands of vesicle-associated proteins from diverse cancer cell Previously, six major protein secretion pathways in Gram-
lines, immune cells, and human fluids including serum, urine, and negative bacteria were known; they can be classified by the
breast milk (Pisitkun, Shen, & Knepper, 2004; Jin et al., 2005; presence or absence of a required signal sequence, Sec (Table 1).
Yates et al., 2005; Admyre et al., 2007; Choi et al., 2007). Sec-dependent pathways include the type II, IV, and V secretion
However, only a few proteomic analyses of bacterial OMVs have systems, which utilize cleavable N-terminal signal peptides
been reported, although they are more ubiquitous and easier to for protein transport across the inner membrane (Kostakioti
obtain than human samples (Post et al., 2005; Bauman & Kuehn, et al., 2005). The type II secretion system, also known as the
2006; Nevot et al., 2006; Lee et al., 2007). These studies did not general secretory pathway, is responsible for the secretion of
achieve high-throughput proteomics and identified only a small several toxins and utilizes the Tat signal motif in addition to Sec
number of well-known proteins, except for E. coli-derived OMVs (Voulhoux et al., 2001). The type IV secretion system allows the
(Lee et al., 2007). In contrast to native OMVs, several proteomic transfer of DNA and multi-subunit toxins, including pertussis
studies have been performed on detergent-extracted OMVs toxin, by conjugation machinery (Cascales & Christie, 2003).
(DOMVs), which are made from whole bacteria with a detergent Depending on the bacterial strain, both Sec-dependent and Sec-
treatment (Nally et al., 2005; Ferrari et al., 2006; Uli et al., 2006; independent secretion have been observed in the type IV system
Vipond et al., 2006). Since outer membrane proteins and LPS of (Desvaux et al., 2004). Proteins using type V secretion, also
OMVs can induce a host immune response, DOMVs from known as the autotransporter pathway, are translocated across
pathogenic strains are promising vaccine candidates. DOMVs the outer membrane via a transmembrane pore formed by a
derived from N. meningitidis are now in clinical trials (Girard self-encoded b-barrel structure (Desvaux, Parham, & Henderson,
et al., 2006). However, although DOMVs are clinically important 2004).
and similar in size and morphology to native OMVs (Ferrari et al., Sec-independent pathways include the type I, III, and
2006), information on DOMV components does not provide any VI secretion systems, which are one-step mechanisms that do not
clue to the biogenesis and functions of native OMVs in bacterial involve periplasmic intermediates (Kostakioti et al., 2005). In the
communities. Therefore, global proteomic studies of native type I secretion system, an ATP-binding cassette transporter-like
OMVs derived from diverse nonpathogenic and pathogenic channel transports various molecules from ions and drugs to
bacteria will help us not only elucidate the biogenesis and proteins (Binet & Wandersman, 1995). The type III system is
functions of OMVs but also develop diagnostics, vaccines, and specific for the transport of factors by pathogenic bacteria and
antibiotics effective against pathogenic strains. allows the direct injection of a protein into a eukaryotic host cell
After an overview of previous biochemical and biological (Galan & Collmer, 1999). Recently, the secretion of several
studies on Gram-negative bacterial OMVs, this review will focus proteins via the type VI pathway was reported in Vibrio cholerae
on current strategies used for proteomic analyses of OMVs, and P. aeruginosa (Mougous et al., 2006; Pukatzki et al., 2006).
emphasizing the impact of those studies on this emerging field.
Finally, future directions for high-throughput and comparative
proteomics studies of OMVs from diverse Gram-negative
B. OMVs as a Novel Protein Secretion Pathway in
bacteria under various environmental conditions will be high-
Gram-Negative Bacteria
lighted in hopes of advancing both basic and clinical sciences.
Protein secretion via OMVs in Gram-negative bacteria has
come of age by defining a distinct type that is independent of
the type I VI secretory systems (Kuehn & Kesty, 2005). The
dynamic feature of Gram-negative cell wall is that it constantly
II. PROTEIN SECRETION IN
discharges OMVs from the cell surface (Fig. 1), which is not
GRAM-NEGATIVE BACTERIA
observed in Gram-positive bacteria (Beveridge, 1999). Growing
evidence suggests that hundreds of proteins, lipids, and genetic
A. Conventional Protein Secretion Pathways in
material might be secreted via OMVs (Dorward, Garon, & Judd,
Gram-Negative Bacteria
1989; Horstman & Kuehn, 2000; Mashburn-Warren & Whiteley,
2006; Lee et al., 2007). For example, export of cytolysin A
Gram-negative bacteria are enclosed within two lipid bilayers,
(ClyA) into the extracellular milieu, which does not follow the
consisting of a phospholipid-rich inner membrane and a
six known protein secretion mechanisms (Wai et al., 2003), is
phopholipid- and LPS-rich outer membrane. The periplasmic
mediated by OMVs (Bendtsen et al., 2005). ClyA is a pore-
space separates these membranes and contains peptidoglycans
forming cytotoxin protein expressed by E. coli and some other
(Beveridge, 1999). In contrast to nucleated eukaryotic cells,
bacterial cytoplasm is not compartmentalized, no large organ- enterobacteria. Therefore, shedding of bacterial OMVs is a novel
protein secretion mechanism in Gram-negative bacteria and
elles are present, and no active transport mechanisms such as
this system performs a variety of important   remote-control 
molecular motors are known (Howard, Rutenberg, & de Vet,
functions for bacterial growth and survival, as well as patho-
2001). However, protein secretion is a basic cellular function
genesis in hosts.
found in all living organisms. Gram-negative bacteria have
536 Mass Spectrometry Reviews DOI 10.1002/mas
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PROTEOMICS IN BACTERIAL OUTER MEMBRANE VESICLES
TABLE 1. Conventional protein secretion pathways in Gram-negative bacteria
III. GRAM-NEGATIVE BACTERIAL OMVS EM studies of Gram-negative bacteria in that era suggested
that secretory vesicles constantly emanate from bacteria under
lysine-limited, phosphate-limited, and normal growth conditions
A. History of Gram-Negative Bacterial OMVs
(Knox, Vesk, & Work, 1966; Mergenhagen, Bladen, & Hsu,
The discovery of OMVs as the universal secretory machinery in
1966; Ingram, Cheng, & Costerton, 1973; Lindsay et al., 1973).
Gram-negative bacteria dates back almost 40 years, owing to the
Moreover, OMVs have been found in every environment in
electron microscope (EM). In the 1960s, active research on the
which Gram-negative bacteria reside, from laboratory cultures,
ultrastructure of bacteria revealed the presence of OMVs and
including planktonic and surface-attached biofilm, to natural
they were named blebs, membranous elements, or channels
environments such as domestic water drains, sewage, and
(Bladen & Waters, 1963; Bayer & Anderson, 1965). Thin section
riverbeds (Beveridge, 1999; Schooling & Beveridge, 2006).
The identification of OMVs from a variety of Gram-negative
strains including E. coli, Veillonella, V. cholerae, P. aeruginosa,
Salmonella typhimurium, and N. meningitidis implies that
virtually all Gram-negative bacteria produce OMVs as an active
and essential process (Bladen & Mergenhagen, 1964; Bayer &
Anderson, 1965; Chatterjee & Das, 1967; Devoe & Gilchrist,
1973; Beveridge, 1999).
B. Current Research Trends on Gram-Negative
Bacterial OMVs
Although bacterial OMVs have long been studied, previous
FIGURE 1. Discharge of OMVs by Gram-negative bacteria. (A) Thin-
reports have focused primarily on pathogenic strains, and many
section EM of E. coli DH5a, showing the formation of OMVs (arrows)
questions remain to be answered before attaining an integrated
on the cell surface. Bar ź 100 nm (B) Magnified EM image of OMVs.
view of the biogenesis and the pathophysiological functions of
The membrane bilayer (arrow) is easily visible. Bar ź 50 nm. [Reprinted
OMVs from both nonpathogenic and pathogenic bacteria (Kuehn
with permission from Proteomics 7: 3143-3153, 2007, Lee EY et al.,
& Kesty, 2005). Recently, biochemical analyses and a few
Global proteomic profiling of native outer membrane vesicles derived
proteomics applications revealed that bacterial OMVs include
from Escherichia coli, with permission from Wiley-VCH Verlag GmbH
proteins, lipids, and genetic material (Kadurugamuwa &
& Co. KGaA. Weinheinm, Germany. Copyright 2007.]
Mass Spectrometry Reviews DOI 10.1002/mas 537
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LEE ET AL.
Beveridge, 1995; Horstman & Kuehn, 2000, 2002; Post et al., and ratios have not been determined. Therefore, systematic
2005; Bauman & Kuehn, 2006; Nevot et al., 2006; Lee et al., studies on the lipid composition of OMVs that might govern
2007). Moreover, genetic studies examining some candidate vesicular fate and function under physiological and pathological
genes that modulate the level of vesiculation suggest possible conditions are a challenge for lipidomics.
biogenesis models for bacterial OMVs (Mashburn-Warren & The presence of DNA within OMVs was identified in N.
Whiteley, 2006; McBroom et al., 2006; McBroom & Kuehn, gonorrhoeae, Haemophilus influenzae, P. aeruginosa, and E. coli
2007). In addition, the pathophysiological roles of OMVs in O157:H7 (Mashburn-Warren & Whiteley, 2006). The fact that
the interspecies world, as well as polymicrobial communities, vesicular DNA is resistant to DNase treatment suggests that it is
are being gradually elucidated. Some groups are studying the present in the lumen of OMVs. Therefore, DNA within OMVs is
physiological relevance of stress responses, including heat-shock expected to be protected from nucleases, thereby increasing the
and antibiotic treatment, which alter the generation of OMVs efficiency of vesicle-mediated DNA delivery into a recipient cell.
(Katsui et al., 1982; Kadurugamuwa & Beveridge, 1995). DNA within OMVs can originate by one of two mechanisms:
In the following subsections, we summarize previous DNA exists in the periplasm, and along with other periplasmic
biochemical and biological research on Gram-negative bacterial components, becomes encapsulated, or DNA in the extracellular
OMVs. environment, potentially derived from lysed bacteria, is incorpo-
rated into OMVs by the   opening and closing  phenomenon
(Renelli et al., 2004). RNA is also a vesicular component,
although in lesser amounts than DNA (Dorward, Garon, & Judd,
1. Components of Gram-Negative Bacterial OMVs
1989).
Although bacterial OMVs were believed to consist of proteins While multiple sources of vesicular components obviously
and lipids from the outer membrane and periplasm but not contribute to the biogenesis and functions of OMVs, the exact
from either the inner membrane or cytoplasmic components vesicular composition of OMVs derived from different bacterial
(Horstman & Kuehn, 2000), growing evidence suggests that strains should not be identical. These differences in vesicular
virulence factors including LPS, cytoplasmic proteins, and components may define unique physiological and pathological
genetic material such as DNA and RNA are components of functions of OMVs derived from specific strains of Gram-
OMVs (Dorward, Garon, & Judd, 1989; Kolling & Matthews, negative bacteria. However, limited data are available on strain-
1999; Ferrari et al., 2006; Lee et al., 2007). specific OMV components, a problem that must be solved in the
The presence of vesicular proteins has been analyzed by near future.
sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS PAGE) with Coomassie or silver staining, as well as
Western blotting with in-house antibodies (Horstman & Kuehn,
2. Biogenesis of Gram-Negative Bacterial OMVs
2000; Ferrari et al., 2006). Several outer membrane proteins
including OmpA, OmpC, and OmpF have been identified, which The mechanisms by which Gram-negative bacteria shed OMVs
represent the most abundant proteins; they have been found in all and sort vesicle-targeted proteins have not been fully determined.
strains of E. coli studied to date (Kesty et al., 2004). Biochemical However, genetic mutant studies, biochemical experiments, and
analysis has detected periplasmic proteins such as alkaline microscopic observations suggest three plausible models for
phosphatase and AcrA in OMVs, supporting the hypothesis that OMV formation, as shown in Figure 2 (Mashburn-Warren &
some periplasmic proteins are sorted into OMVs by encapsula- Whiteley, 2006). The first model suggests that vesicles are
tion during vesicle formation (Horstman & Kuehn, 2000). generated by the loss of cell envelope integrity that occurs when
Pathogenic bacteria secrete OMVs that contain several the outer membrane expands more quickly than the underlying
virulence factors, including toxins, adhesins, invasins, and other peptidoglycan layer (Wensink & Witholt, 1981). The second
related enzymes (Kesty & Kuehn, 2004; Kesty et al., 2004; model is that the formation of OMVs is linked to the turgor
Kuehn & Kesty, 2005). Toxins are the best characterized factors pressure of the cell envelope, which changes with the
that might be involved in OMV-mediated bacterial pathogenesis. accumulation of peptidoglycan fragments in the periplasm (Zhou
For example, LPS, which are critical components of all et al., 1998). A recent study in P. aeruginosa proposed the third
Gram-negative bacteria, can activate host immune responses model, in which quinolone signal molecules enhance the anionic
via production of various cytokines. The large number of repulsion between LPS by destabilizing the Mg2þ and Ca2þ salt
vesicular toxins that contribute to the pathogenesis of infection bridges in the outer membrane, thereby causing membrane
was summarized in a recent report (Kuehn & Kesty, 2005). The blebbing (Mashburn & Whiteley, 2005). These three mechanisms
ability of OMVs to adhere to and invade host cells via adhesins may not be mutually exclusive and may contribute collectively to
and invasins is important in initiating vesicle-mediated patho- the biogenesis of bacterial OMVs. Further study is needed, since
genesis. For example, Ail, IpaB, IpaC, and IpaD play important recent studies suggest that OMV production is independent of
roles in interactions with and invasion of host cells (Kaduruga- membrane instability (McBroom et al., 2006) and controversial
muwa & Beveridge, 1998; Kesty & Kuehn, 2004). results have come from the mutation of lipoproteins (Bernadac
Thin layer chromatography revealed the presence of et al., 1998; Rolhion et al., 2005).
glycerophospholipids, phosphatidlyethanolamine, phosphatidyl- Currently, little is known about the mechanisms by which
glycerol, and cardiolipin in enterotoxigenic E. coli-derived bacteria sort proteins into OMVs. However, analysis of vesicular
OMVs (Horstman & Kuehn, 2000). The lipid profiles of OMVs proteins by SDS PAGE have shown different banding patterns in
are similar to those of the outer membrane, but their specific types the OMVs compared to the outer membrane, periplasm, and other
538 Mass Spectrometry Reviews DOI 10.1002/mas
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PROTEOMICS IN BACTERIAL OUTER MEMBRANE VESICLES
FIGURE 3. Proteins present in OMVs derived from E. coli DH5a.
Coomassie blue-stained SDS PAGE comparison of the proteins from
whole-cell lysates (WC), periplasmic proteins (PP), outer membrane
proteins (OMP), and OMVs showing specific protein sorting into
vesicles. Molecular weight standards are indicated on the left (kDa).
[Reprinted with permission from Proteomics 7: 3143 3153, 2007, Lee
EY et al., Global proteomic profiling of native outer membrane vesicles
derived from Escherichia coli, with permission from Wiley-VCH Verlag
GmbH & Co. KGaA. Copyright 2007.]
OMVs that are shed into the extracellular milieu. Budding of
OMVs from the limiting membrane can be inferred from human
cell-derived microvesicles that are reportedly associated with
specific membrane sites called lipid rafts (Rajendran & Simons,
2005).
3. Physiological and Pathological Functions of
Gram-Negative Bacterial OMVs
Since OMVs bear diverse proteins, LPS, outer membrane lipids,
genetic material, other factors associated with virulence, and
FIGURE 2. Proposed models for biogenesis of Gram-negative bacterial
conjugational machinery to target other bacteria or host cells,
OMVs. Model 1: OMVs are liberated from specific regions on the cell
they should play diverse roles as intercellular communicasomes
surface where peptidoglycan-associated lipoproteins are missing due
not only in bacterial communities but also in interspecies worlds.
to the faster expansion of the outer membrane than the underlying
Gram-negative bacterial OMVs might play a role in the transfer
peptidoglycan layer. Model 2: Accumulation of peptidoglycan frag-
of proteins and genetic material between bacterial cells, in the
ments in the periplasm causes increased turgor pressure, thereby increas-
elimination of competing organisms, in cell-to-cell signaling and
ing blebbing of the outer membrane. Model 3: In the P. aeruginosa outer
membrane, PQS sequesters the positive charge of Mg2þ, which results in bacterial survival, and in the delivery of toxins to host cells
enhanced anionic repulsion between LPS molecules and membrane
(Kuehn & Kesty, 2005; Mashburn-Warren & Whiteley, 2006).
blebbing. OM, outer membrane; PG, peptidoglycan; IM, inner mem-
OMVs are involved in the transfer of proteins as well as
brane; PQS, Pseudomonas quinolone signal. [Reprinted with permission
genetic material in polymicrobial communities. OMVs derived
from Molecular Microbiology 61: 839 846, 2006, Mashburn-Warren
from P. aeruginosa showed beneficial effects to their own group
and Whiteley, Special delivery: vesicle trafficking in prokaryotes, with
by transferring an antibiotic resistance protein, b-lactamase,
permission from Blackwell Publishing Ltd Oxford, UK. Copyright
to increase survival (Mashburn-Warren & Whiteley, 2006).
2006.]
Predatory roles of OMVs have been proposed, in which E. coli-
and P. aeruginosa-derived vesicles can kill competing bacteria by
cellular fractions, suggesting that specific protein sorting peptidoglycan degradation or cell lysis via vesicular components
mechanisms are in effect when OMVs are produced (Fig. 3) such as murein hydrolases (Kadurugamuwa & Beveridge, 1996;
(Horstman & Kuehn, 2000; Lee et al., 2007). Although further Li, Clarke, & Beveridge, 1998). Furthermore, OMVs package
study of this issue is needed, these findings imply the presence of chromosomal, plasmid, and phage DNA as well as RNA, which
  hot spots  for vesicle budding: the outer membrane becomes may increase genetic diversity by transforming neighboring
loosely attached to the bacterium at specific sites and forms bacteria (Kuehn & Kesty, 2005).
Mass Spectrometry Reviews DOI 10.1002/mas 539
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LEE ET AL.
Bacterial OMVs also play protective roles that contribute to DOMVs are produced under artificial conditions, DOMVs and
bacterial survival by reducing toxic compounds and antibiotics, native OMVs may have different protein components. As shown
and by facilitating the release of attacking phages (Loeb & in Table 2, a few studies have been published regarding the
Kilner, 1978; Kobayashi et al., 2000). Recently, McBroom proteomics of native OMVs. These include OMVs isolated from
and Kuehn (2007) performed a genetic mutant study under pathogenic bacteria such as N. meningitidis and P. aeruginosa
stress conditions by treatment with 10% ethanol or an outer (Post et al., 2005; Bauman & Kuehn, 2006), nonpathogenic
membrane-damaging antimicrobial peptide and found that over- bacteria including Pseudoalteromonas antarctica NF3, and
vesiculating mutant strains had enhanced survival due to the E. coli (Nevot et al., 2006; Lee et al., 2007), and peculiar mutant
release of misfolded proteins via increased shedding of OMVs forms of N. meningitidis and extraintestinal pathogenic E. coli
(McBroom & Kuehn, 2007). (Ferrari et al., 2006; Berlanda Scorza et al., 2007).
Some bacteria have coevolved in a symbiotic relationship In the following subsections, we summarize previous
with their hosts, although infectious pathogens may cause some proteomic studies on native OMVs derived from Gram-negative
problems in hosts. Therefore, examining the roles of OMVs bacteria and discuss in detail clues provided by proteomics that
in the interspecies community is important to understanding elucidate the biogenesis and functions of bacterial OMVs.
the mechanisms of symbiosis and pathogenesis. Diverse Gram-
negative pathogens have exploited potent virulence strategies by
vesicle-mediated toxin delivery to host cells (Kuehn & Kesty,
A. Preparation of Gram-Negative Bacterial OMVs
2005). In the case of enterotoxigenic E. coli-derived heat-labile
enterotoxins (LT), most ( 95%) are secreted via OMVs (Horst- Efficient OMV preparation without any contamination by non-
man & Kuehn, 2002). Some toxins, including LTand leukotoxin, vesicular components is a critical prerequisite for proteomic
are more active when they are associated with vesicles rather than analysis. Generally, bacterial OMVs are isolated from the culture
in a free form (Kuehn & Kesty, 2005). Moreover, LPS and the supernatant using a combination of differential centrifugation
outer membrane proteins present in vesicles can activate host to remove cells and cell debris, and ultracentrifugation to pellet
immune responses via Toll-like receptors. These LPS and the OMVs (Wai et al., 2003). However, ultracentrifugation
surface-localized antigens from pathogenic bacteria-derived alone does not discriminate between OMVs and other membrane
vesicles can cause overstimulated inflammatory responses or debris or large protein aggregates. Filtration of the cell-culture
septic shock in hosts (Namork & Brandtzaeg, 2002). supernatant through 0.22 0.45 mm filters before ultracentrifu-
gation may reduce contamination (Horstman & Kuehn, 2000;
Ferrari et al., 2006; Berlanda Scorza et al., 2007). Recent progress
in the biology of OMVs shows that density gradient centrifuga-
tion is one of the best separation methods to remove OMVs from
IV. CURRENT PROTEOMICS IN GRAM-NEGATIVE
contaminating protein aggregates, pili, and flagella (Bauman &
BACTERIAL OMVS
Kuehn, 2006; Lee et al., 2007). Gel filtration chromatography
Although previous biochemical, biological, and genetic studies is an alternative method to isolate OMVs with high purity.
help us to understand the vesicular components, biogenesis, The beads in a gel filtration chromatography column contain
and diverse roles of OMVs in polyspecies communities, the pores that can fractionate vesicles on the basis of differential
information provided by those studies does not afford a com- diffusion and size exclusion. Post et al. (2005) purified
prehensive understanding of the emerging biology of bacterial OMVs from N. meningitidis using a Sephacryl S500 column
OMVs. MS-based proteomic studies offer a powerful way to (Post et al., 2005). Gel filtration chromatography is an effective
clarify the mechanisms of vesicle formation and the pathophy- method for purifying OMVs that are relatively homogeneous
siological roles of OMVs by drawing a global map of diverse in size.
bacterial OMVs. Recently, we reported the proteomic profile of native OMVs
Proteomic studies have been successfully used to study derived from representative strains of nonpathogenic E. coli
whole cellular proteins from diverse bacteria and to study DH5a (Lee et al., 2007). With some modifications of previously
bacterial adaptation to various stress situations (Washburn & described purification methods (Horstman & Kuehn, 2000; Wai
Yates, 2000; Hecker & Volker, 2004; Bandow & Hecker, 2007). et al., 2003; Rolhion et al., 2005), we obtained highly pure
However, complexity at the whole-cell level has a limited ability OMVs secreted by DH5a cells using two sequential steps
to provide systematic insights into cell biology and often fails (Fig. 4A). In the first step, OMVs were isolated from the culture
to identify low-abundance proteins, including outer membrane supernatant using a combination of differential centrifugation to
proteins, which are inevitably masked by high-abundance remove cells and cell debris; filtration through a 0.45 mm filter;
proteins (Brunet et al., 2003). Because OMVs may represent pre-centrifugation at 20,000g and 40,000g to remove any large
nano-sized extracellular organelles of bacteria, the application vesicles, vesicle aggregates, and cell debris; and then ultra-
of organelle proteomics to bacterial OMVs will define new centrifugation at 150,000g. In the second purification step, the
biological processes for interactions among bacterial cells, enriched OMVs were further purified using sucrose density
symbiosis, and pathogenesis in hosts. gradients to remove any remaining contaminants. EM of purified
Regardless of the importance of native OMVs, which may OMVs revealed that almost all were small, closed vesicles
act as key mediators for intercellular communications, previous ranging from 20 to 40 nm in diameter, and no membrane whorls,
proteomic research has focused on DOMVs (Nally et al., 2005; fragments of lysed vesicles, large vesicles, or pili were detected
Ferrari et al., 2006; Uli et al., 2006; Vipond et al., 2006). Since (Fig. 4B).
540 Mass Spectrometry Reviews DOI 10.1002/mas
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PROTEOMICS IN BACTERIAL OUTER MEMBRANE VESICLES
TABLE 2. Summary of proteomic studies on native bacterial OMVs
B. Proteomic Analysis of Gram-Negative protein separation. However, the limitations of this approach
for membrane proteins are well-known. The major obstacle
Bacterial OMVs
is poor solubility of membrane proteins in the non-detergent
In addition to the purity of OMVs, reduction of proteome isoelectric focusing buffer that causes the precipitation of
complexity by protein separation is the key step to identifying a proteins at their isoelectric points (Wu & Yates, 2003).
large number of vesicular proteins via proteomic analysis. Two- Moreover, 2-DE cannot properly resolve high molecular
dimensional gel electrophoresis (2-DE) is a powerful tool for weight, very basic, or hydrophobic proteins (Wu & Yates,
FIGURE 4. Methods of preparation for OMVs derived from E. coli DH5a. A: Procedure for preparing
OMVs from DH5a. B: Negative-staining transmission EM of purified OMVs after sucrose density gradient
centrifugation, showing a homogeneous size of 20 40 nm. Bar ź 50 nm. [Reprinted with permission from
Proteomics 7: 3143 3153, 2007, Lee EY et al., Global proteomic profiling of native outer membrane
vesicles derived from Escherichia coli, with permission from Wiley-VCH Verlag GmbH & Co. KGaA.
Copyright 2007.]
Mass Spectrometry Reviews DOI 10.1002/mas 541
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LEE ET AL.
2003). The fact that outer membrane proteins, the major one proteins (DegQ/SurA), and motility proteins related to
components of OMVs, are highly basic implies that major fimbriae (FliC) or pilus (PilQ). These conserved vesicular
components of vesicular proteins are incompletely resolved proteins provide an integrated view of the biogenesis and
in 2-DE (Post et al., 2005). function of OMVs in nonpathogenic and pathogenic bacteria,
The combination of one-dimensional (1-D) SDS PAGE which will be discussed in the following subsections. For
and liquid chromatography (LC) MS/MS provides a powerful pathogenic strains, virulence factors including hemolysin, IgA
alternative to 2-DE-based proteomic analysis (Aebersold & protease, and macrophage infectivity potentiator were also
Mann, 2003). Although 1-D SDS PAGE can efficiently identified (Post et al., 2005; Ferrari et al., 2006).
separate proteins, even membrane proteins, the limitation of Many researchers believe that OMVs are composed solely of
this approach in high-throughput mass analysis is the increased outer membrane and periplasmic proteins, whereas cytoplasmic
protein complexity in each gel fraction. This problem can proteins are excluded (Horstman & Kuehn, 2000). However,
easily be overcome by using LC to separate the extracted although it is still debated, proteomic analyses have shown that
peptides based on hydrophobicity. Several groups have used this native OMVs and DOMVs contain cytoplasmic proteins as well
strategy (Post et al., 2005; Nevot et al., 2006), but they only (Molloy et al., 2000; Henry et al., 2004; Ferrari et al., 2006; Wei
examined prominent protein bands of interest from gels, which et al., 2006; Xu et al., 2006; Lee et al., 2007). Among vesicle-
might result in the identification of less than 50 vesicular associated cytoplasmic proteins, highly abundant proteins like
proteins because of missing the less abundant and unknown EF-Tu, GroEL, DnaK, and two ribosomal proteins (S1 and
proteins (Table 2). Because the molecular weights of vesicular L7/12) have also been detected from cell supernatants or outer
proteins are different and OMVs also contain less abundant membrane fractions (Ferrari et al., 2006). Moreover, the fact that
proteins, our group separated vesicular proteins by 1-D SDS vesicles carry DNA and RNA, and that translation of outer
PAGE, cut the gel into five slices of equal size, and subjected it membrane proteins might occur simultaneously with their
to trypsin digestion. From two independent nano-LC electro- integration into the membrane, suggest that transcriptional and
spray ionization (ESI) MS/MS analyses of the extracted ribosomal proteins can be sorted into vesicles during the
peptides, we identified 2,606 and 2,816 proteins with high- informational process (Dorward, Garon, & Judd, 1989; Kadur-
confidence peptide sequences, with an error rate less than 1% (F ugamuwa & Beveridge, 1995; Kolling & Matthews, 1999; Yaron
score > 2.17). Since peptides that are shared by multiple et al., 2000). Determining whether cytoplasmic proteins are
proteins are less informative than unique peptides, we used indeed components of native OMVs should be a goal of future
the protein hit score (PHS) for reliable protein identification studies.
(Park et al., 2006). Our analysis showed that proteins with
PHS > 1 were identified by multiple peptides that are unique
and shared with only a few proteins. Using a highly stringent
D. Proteins Involved in Biogenesis of Gram-Negative
filter allowing only proteins with PHS > 1 that were filtered
Bacterial OMVs
again to reduce any repeated or homologous proteins, we finally
identified a total of 141 proteins, including 127 previously Although the mechanism of OMV formation has not yet been
unknown vesicular proteins, with high confidence, and reprodu- elucidated, several vesicular proteins identified by proteomic
cibility (Lee et al., 2007). analyses support the first and second models (Fig. 2). Omps, Tol-
Pal, YbgF, and Lpp lipoproteins found in the OMV proteome
should be involved in outer membrane integrity and might
help liberate OMVs from the bacterial cell surface by initiating
C. Proteins Identified by Proteomic Analyses of
faster expansion of the outer membrane than the underlying
Gram-Negative Bacterial OMVs
peptidoglycan layer (Bernadac et al., 1998). Related to
The available proteomic studies on bacterial OMVs have defined the second model, murein hydrolases, including MltA, MipA,
more than 200 vesicular proteins from four native bacterial and MltE, and SLP, may lead to the accumulation of peptidoglycan
two mutant strains (Table 2) (Post et al., 2005; Bauman & fragments in the periplasmic space, resulting in increased turgor
Kuehn, 2006; Ferrari et al., 2006; Nevot et al., 2006; Berlanda pressure and causing the discharge of OMVs (Lommatzsch
Scorza et al., 2007; Lee et al., 2007). Although the names of et al., 1997).
bacterial proteins are different in each species, they can be When OMV proteomes are annotated according to their
classified into protein families based on both their sequence subcellular distribution, OMVs are highly enriched in outer
homology and function. When the identified vesicular proteins membrane and periplasmic proteins, whereas inner membrane
were categorized by protein family, several protein families proteins are excluded (Post et al., 2005; Lee et al., 2007).
were common in OMVs derived from several species of Gram- For example, of 141 proteins identified in E. coli-derived OMVs,
negative bacteria (Table 3). Porins (Omps, PorA, PorB, and 65 (46.1%), 16 (11.3%), 7 (5.0%), 52 (36.9%), and 1 (0.7%)
OprF), abundant outer membrane proteins, are found in most proteins were derived from the outer membrane, periplasm, inner
OMVs. Murein hydrolases (Mlt and SLT) are responsible for the membrane, cytoplasm, and extracellular space, respectively
hydrolysis of certain cell wall glycopeptides, particularly (Fig. 5) (Lee et al., 2007). In contrast, from the EchoBASE
peptidoglycans. Multidrug efflux pumps (Mtr, Mex, and TolC) database of all 4,345 E. coli proteins, 149 (3.4%), 350 (8.1%),
function in the release of toxic compounds (Kobayashi et al., 974 (22.4%), 2,862 (65.9%), and 10 (0.2%) are distributed in
2000). Moreover, most OMVs derived from different strains the outer membrane, periplasm, inner membrane, cytoplasm, and
contain ABC transporters (LamB and FadL), protease/chaper- extracellular space, respectively, suggesting that outer membrane
542 Mass Spectrometry Reviews DOI 10.1002/mas
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PROTEOMICS IN BACTERIAL OUTER MEMBRANE VESICLES
TABLE 3. Protein families identified by proteomic analyses of Gram-negative bacterial OMVs
(Continued )
Mass Spectrometry Reviews DOI 10.1002/mas 543
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LEE ET AL.
TABLE 3. (Continued )
(Continued )
544 Mass Spectrometry Reviews DOI 10.1002/mas
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PROTEOMICS IN BACTERIAL OUTER MEMBRANE VESICLES
TABLE 3. (Continued )
a
Accession numbers of individual proteins originate from each reference.
and periplasmic proteins are more commonly sorted into OMVs hypothesis that special sorting mechanisms are in effect and/or
(Misra et al., 2005). Moreover, the inclusion of particular proteins the OMVs bud at specific vesiculation sites.
in OMVs does not appear to be a strict function of their
abundance. As shown in Table 4, several low-abundance outer
membrane proteins, including FimD, FecA, FhuE, and FepA,
E. Proteins Involved in Biological Functions of
and periplasmic proteins, including YddB, SLT, MalM, and
Gram-Negative Bacterial OMVs
PRC, were identified, whereas the most abundantly expressed
periplasmic proteins, such as OppA, FimA, HdeA, and LivJ, were In addition to supporting previously known biological roles of
not (Corbin et al., 2003). These results further support the OMVs, vesicular proteins identified in proteomic studies suggest
Mass Spectrometry Reviews DOI 10.1002/mas 545
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LEE ET AL.
inorganic ions (FepA, FhuA, and Fiu), and nucleosides (Tsx)
exert their roles as delivery systems in bacterial communities.
In particular, TonB-dependent receptors (BtuB, FhuA, and FhuE)
in OMVs have been suggested to be nutrient sensors and
transporters, and their presence has been postulated to represent
an alternative mechanism for survival in nutrient-limited systems
(Nevot et al., 2006). Furthermore, murein hydrolases (e.g., MltA,
SLT) in OMVs are involved not only in OMV biogenesis as
described above, but also in predatory activities by killing
competing bacteria via cell wall degradation (Kadurugamuwa &
Beveridge, 1996; Li, Clarke, & Beveridge, 1998).
In the host environment, effective pathological functions of
OMVs can be achieved by increased resistance to bactericidal
factors. OmpT in vesicles may degrade cationic antimicrobial
peptides produced by epithelial cells or macrophages, and Iss
may increase serum survival of OMVs (Stumpe et al., 1998;
Nolan et al., 2003). Moreover, in addition to pathogenic-specific
FIGURE 5. Subcellular distribution of vesicular and cellular proteins
toxins, outer membrane porin proteins, including OmpA and
present in E. coli. When compared to the complete E. coli proteome,
OmpF, which are enriched in OMVs, have immunostimulatory
DH5a-derived OMVs are highly enriched in outer membrane proteins,
activity and induce leuckocyte migration (Galdiero et al., 1999).
whereas inner membrane proteins are excluded. [Adapted from Lee
Pathogenic-specific adherent/invasive proteins and outer mem-
et al. (2007).]
brane porins, including OmpA, OmpW, and OmpX, are involved
in targeting OMVs to host cells. Furthermore, OmpA enhances
the uptake of LPS by macrophages and contributes to the invasion
novel functions, and we can specify what proteins are involved
of brain microvascular endothelial cells (Korn et al., 1995;
in each physiological and pathological function. As shown in
Prasadarao et al., 1996).
Figure 6, protein function can be largely classified by the
interacting partner, including bacteria and host cells. First, the
organic solvent tolerance protein (OstA), multidrug-resistant
efflux pumps (Mex, Mtr, and TolC), and phage target receptors
V. FUTURE DIRECTIONS FOR PROTEOMIC
(FepA, LamB, and OmpA) in E. coli, N. meningitidis, and
STUDIES IN GRAM-NEGATIVE BACTERIAL OMVS
P. antarctica NF3-derived OMVs may contribute to bacterial
survival by reducing levels of toxic compounds such as n-hexane Previous proteomic studies have established a proteome database
and antibiotics, and by facilitating the release of attacking phages of OMVs derived from four native and two mutant bacteria.
(Post et al., 2005; Nevot et al., 2006; Lee et al., 2007). ABC These studies have provided important information about the bio-
transporters for specific nutrients (LamB, BtuB, and FadL), genesis, pathophysiological functions, and protein composition
TABLE 4. Sorting profiles of vesicular proteins in E. coli DH5a
OMP, outer membrane protein; PP, periplasmic protein.
a
Abundance represents mRNA signal intensity on GeneChip.
546 Mass Spectrometry Reviews DOI 10.1002/mas
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PROTEOMICS IN BACTERIAL OUTER MEMBRANE VESICLES
FIGURE 6. Proposed physiological and pathological functions of Gram-negative bacterial OMVs.
Functions of Gram-negative bacterial OMVs are predicted based on the available proteomes of OMVs
derived from nonpathogenic and pathogenic bacteria. [Color figure can be viewed in the online issue, which
is available at www.interscience.wiley.com.]
of bacterial OMVs (Post et al., 2005; Bauman & Kuehn, 2006; reactions suggest that bacterial OMVs are involved in the
Ferrari et al., 2006; Nevot et al., 2006; Berlanda Scorza et al., progress of septic shock. Therefore, proteomic analysis of OMVs
2007; Lee et al., 2007). However, compared to human micro- obtained from the serum of septic human patients or septic rats
vesicles, for which significant progress has been made in defining should increase our understanding about the pathological roles of
the proteomes of vesicular components (Yates et al., 2005), OMVs in septic shock (Brandtzaeg et al., 1992; Hellman et al.,
proteomic information on bacterial OMVs still remains scarce 2000). Moreover, vesicles shed by N. meningitidis have been
except for E. coli (Berlanda Scorza et al., 2007; Lee et al., 2007). found in the cerebrospinal fluid and blood of a patient with
Limited proteomic data make it difficult to elucidate the exact meningitis (Stephens et al., 1982; Brandtzaeg et al., 1992;
mechanism of vesicle formation and new functions of OMVs. Namork & Brandtzaeg, 2002). In the case of Borrelia
Further studies are therefore necessary to construct a vesicular burgdorferi-infected mice, OMVs were detected in the urine
proteome for diverse Gram-negative bacteria under a variety of and blood (Dorward, Schwan, & Garon, 1991). Furthermore,
conditions. Overall, the future directions for proteomic analyses OMVs derived from Bacteroides (Porphyromonas) gingivalis,
on Gram-negative bacterial OMVs are summarized in Figure 7. which causes oral cavities, inflammation, and bleeding at
peritonitis sites, can be obtained from dental plaque samples
(Grenier & Mayrand, 1987; Imamura et al., 1995).
A. OMVs Derived from Diverse
Gram-Negative Bacteria
B. Preparation of Gram-Negative Bacterial OMVs
Diverse Gram-negative bacteria provide unlimited material for
OMVs, as most have been reported to secrete vesicles. Valuable As noted above, OMVs are usually prepared by ultracentrifuga-
samples can be obtained from host fluids and tissues (Table 5). tion followed by density gradient centrifugation or gel filtration
The facts that Gram-negative bacteria are the main causes of (Horstman & Kuehn, 2000; Wai et al., 2003; Post et al., 2005; Lee
septic shock, and that LPS and outer membrane proteins of et al., 2007). Other methods such as free-flow electrophoresis
bacterial OMVs elicit a complex pattern of inflammatory (FFE) and capillary electrophoresis (CE) can be used to isolate
Mass Spectrometry Reviews DOI 10.1002/mas 547
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LEE ET AL.
FIGURE 7. Future directions for high-throughput and comparative proteomics in OMVs originating from
diverse Gram-negative bacteria under various environmental conditions.
OMVs in intact form and with high purity. FFE is a powerful tool purity by discriminating similar-density membrane fragments,
to separate human cellular organelles such as peroxisomes, which are difficult to remove solely by density gradient
lysosomes, endosomes, melanosomes, and Golgi vesicles, as ultracentrifugation.
well as mitochondria (Morre, Morre, & Heidrich, 1983; Fuchs, CE is an analytical technique that employs narrow
Male, & Mellman, 1989; Marsh, 1989; Kushimoto et al., 2001; capillaries for electric field-mediated separation of particles with
Mohr & Volkl, 2002; Zischka et al., 2006). This system allows a surface charge. Recently, CE was used to separate human
purification of bacterial OMVs in a native state and with high organelles including mitochondria, acidic organelles, nuclei, and
548 Mass Spectrometry Reviews DOI 10.1002/mas
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PROTEOMICS IN BACTERIAL OUTER MEMBRANE VESICLES
TABLE 5. OMVs found in host fluids and tissues
lipid vesicles (Gunasekera, Musier-Forsyth, & Arriaga, 2002; extreme pI, integral membrane proteins, and low-abundance
Duffy et al., 2002; Fuller & Arriaga, 2003; Owen, Strasters, & proteins (Graham, Graham, & McMullan, 2007).
Breyer, 2005). The electrophoretic mobility of organelles is Furthermore, a combination of matrix-assisted laser
determined by the electrical charge on the surface of organelles, desorption/ionization time-of-flight (MALDI-TOF) and ESI
their morphology, and size (Owen, Strasters, & Breyer, 2005). mass spectrometer can also increase coverage and the number
Using this technique, a very small number of organelles can be of identified proteins. ESI-MS/MS and MALDI-TOF-MS
separated electrokinetically or hydrodynamically into a capillary analyses of the same sample usually identify different sets of
(Fuller & Arriaga, 2004). Therefore, CE should be used to proteins (Bodnar et al., 2003). Therefore, high proteome
prepare OMVs with high purity. coverage for OMVs can be achieved by the combination of
multidimensional protein and/or peptide fractionation methods,
as well as by the combination of various MS methods.
C. Strategies for the Proteomic Analysis of
Gram-Negative Bacterial OMVs
2. Bioinformatics for Annotation of Vesicular Proteins
To clarify how bacteria shed vesicles and identify the
1. Protein Identification
physiological and pathological functions of OMVs, annotation
For a comprehensive proteomic analysis of OMVs derived from of identified vesicular proteins based on subcellular localization
various Gram-negative bacteria, extensive prefractionation of and function is important. Previous proteomic analyses of OMVs
samples on the protein and/or peptide levels before mass analysis showed that vesicular proteins are derived from various
should be considered. Although 2-DE is a powerful tool for subcellular locations in bacteria (Post et al., 2005; Ferrari et al.,
protein separation, this method cannot properly resolve high- 2006; Lee et al., 2007). Annotation of vesicular proteins by
molecular-weight, very basic, or hydrophobic proteins (Wu & subcellular localization in bacteria can help us elucidate the
Yates, 2003), suggesting that protein separation by 2-DE may mechanism of OMV biogenesis, as well as identify possible
be not suitable for a global proteomic analysis of OMVs. contaminants in proteomic analyses. Currently, several bio-
Combination of 1-D SDS PAGE and LC MS/MS provides a informatics tools are available for bacterial protein localization
powerful alternative to 2-DE-based proteomic analysis, as noted including PSORTb (Gardy et al., 2005), EchoBASE (Misra et al.,
above (Aebersold & Mann, 2003). Alternatively, multidimen- 2005), Proteome Analyst (Lu et al., 2004), and SubLoc (Hua &
sional protein identification technology (MudPIT) (Washburn, Sun, 2001). They predict the localization of bacterial proteins on
Wolters, & Yates, 2001) should be useful for proteome analysis of the basis of known motifs or cleavage sites. Furthermore, the
OMVs. Because MudPIT separates peptides using a combination computational prediction of subcellular localization of vesicular
of two different kinds of LC prior to MS analysis, this system proteins offers numerous insights that, for example, can assist
greatly reduces the complexity of the proteome at the peptide in functional analyses of OMVs.
level, resulting in the identification a large number of proteins. For the functional analysis of vesicular proteins, several
Using this process, 1,484 proteins were identified from bioinformatics tools are available (Ouzounis et al., 2003). One
Saccharomyces cerevisiae (Washburn, Wolters, & Yates, 2001). of the most influential classification schemes comes from a
Furthermore, MudPIT is suitable for identifying proteins with hierarchy of properties for the gene products of E. coli, which was
Mass Spectrometry Reviews DOI 10.1002/mas 549
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LEE ET AL.
later extended to develop multifunctional classes (Riley, 1993; technology, it is still not technically feasible to obtain a complete
Serres & Riley, 2000). Inspired by this classification, the expression profile from a single two-dimensional gel because
automatic genome-annotation system GeneQuiz was developed some proteins are not well separated, such as those that
based on the keyword mapping of protein families to 14 are extremely basic or acidic, small or large, or of low abundance
functional classes (Andrade et al., 1999). Gene ontology (Wu & Yates, 2003).
classification, which comprises the three categories of molecular The second method for comparative proteomics is based on
function, biological process, and cellular component, is com- stable isotope tagging of proteins and automated LC MS/MS
monly used for protein annotation (Ashburner et al., 2000). The analysis of peptides derived from complex protein mixtures
Kyoto Encyclopedia of Genes and Genomes uses a different (Conrads et al., 2001). Isotope-coded affinity tags (ICATs) and
method of classifying genes and proteins by their participation isobaric tags for relative and absolute quantification (iTRAQ) are
in or association with metabolic pathways (Kanehisa et al., commonly used chemical isotopic labeling strategies that
2002). Since each data mining method uses a different strategy to differentially label sulfhydryls or primary amines of proteins or
predict the function of bacterial proteins, a combination of peptides, respectively (Gygi et al., 2002; Choe et al., 2005). Using
bioinformatics tools should be used to annotate the identified ICATs, several comparative analyses of bacterial proteomes have
vesicular proteins to elucidate the numerous physiological and been reported, including the P. aeruginosa proteome during
pathological functions of Gram-negative bacterial OMVs. anaerobic growth (Peng et al., 2005) and magnesium-limited
conditions (Guina et al., 2003). The major disadvantage of this
technique is that cysteine is a relatively rare amino acid that is not
present in 10 20% of bacterial proteins (Cordwell, 2006). Since
D. Comparative Proteomics in Gram-Negative
all proteins and peptides have primary amines on their N-terminal
Bacterial OMVs
amino acids or lysine, iTRAQ should be better for comparative
In contrast to the static nature of the genome sequence, which proteomics than using ICATs (Danielsen et al., 2007).
provides the blueprint for all protein-based cellular building Another widely applicable technique is stable-isotope
blocks, the proteome is highly dynamic (Bandow & Hecker, labeling of amino acids in cell culture (SILAC) (Mann, 2006;
2007). The protein composition of bacteria is constantly adjusted Ong et al., 2002). In the SILAC procedure, cells are grown in the
to facilitate survival, growth, and reproduction in an ever- presence of an isotopically heavy amino acid for several
changing environment. Bacteria face highly variable growth generations, thereby replacing essentially all of the naturally
conditions and stress situations with respect to temperature, pH, occurring light amino acid in all proteins (Gingras et al., 2007).
osmolarity, nutrient availability, and host infection, among other The advantage of SILAC over ICATs and iTRAQ is that SILAC
factors. Like the whole bacteria proteome, components of OMVs can efficiently label all proteins present in complex samples,
are influenced by environmental stress and bacterial status whereas the labeling efficiency of ICATs and ITRAQ may depend
(Katsui et al., 1982; Horstman & Kuehn, 2002; McBroom & on several factors, including the status of proteins (i.e., native vs.
Kuehn, 2007). Some vesicular proteins are temporarily expressed, denatured) (Gruhler et al., 2005). SILAC has been successfully
whereas other proteins are expressed after signal transduction used in several recent comparative proteomic studies, including
from the extracellular milieu, or at certain bacterial growth profiling of the dynamic association of chaperonin-dependent
phases (Kuehn & Kesty, 2005). Moreover, the release of OMVs protein folding in E. coli (Kerner et al., 2005).
increases when bacteria are exposed to conditions such as
high temperature, exposure to antibiotics or serum, or nutrient
deprivation (Post et al., 2005). Therefore, comparative proteomic
analyses of OMVs derived from diverse conditions are expected
VI. APPLICATIONS OF PROTEOMIC STUDIES ON
to provide new functional insights into bacterial OMV biology,
GRAM-NEGATIVE BACTERIAL OMVS
facilitate the identification of pathogenic markers, and contribute
to the discovery of proteins as therapeutic targets. As described above, high-throughput and comparative proteo-
Three approaches have been most commonly used to mics on bacterial OMVs will decipher the mechanisms under-
generate quantitative profiles of complex protein mixtures lying intercellular communication by elucidating the biogenesis
(Zhang, Yan, & Aebersold, 2004). The first is a combination of and specialized functions of bacterial OMVs. In addition to
2-DE and MS (Aebersold & Mann, 2003). Classical 2-DE is still improving our understanding of the basic biology of bacterial
the method of choice for quantitative analysis of the proteome, OMVs, knowledge about the vesicular proteome can facilitate a
and difference gel electrophoresis (DIGE) is a commonly used variety of biotechnology applications of OMVs, especially in
comparative 2-DE technique (Marouga, David, & Hawkins, human medical research, by identifying specific biomarkers that
2005). In DIGE, proteins from different samples are labeled with can be used to develop diagnostic and therapeutic tools against
different fluorescent dyes, mixed equally, and resolved by 2-DE. pathogenic organisms. Because bacterial OMVs can be dissemi-
The protein samples are visualized using fluorescence imaging to nated from infectious sites and circulated in fluids within the host
enable detection of differences in protein abundance between (Brandtzaeg et al., 1992; Hellman et al., 2000), characterization
samples, and the proteins that differ in abundance can be of vesicles from patients with bacterial-associated disease
identified by MS (Marouga, David, & Hawkins, 2005). Using symptoms is an effective way to diagnose pathogenesis.
DIGE, comparative proteomic studies were carried out on Moreover, particular interest has emerged in the use of
DOMVs and OMVs derived from N. meningitidis (Ferrari OMVs as vehicles for stimulation of host immune responses.
et al., 2006). However, in spite of the progress in 2-DE These studies have already led to clinical trials with DOMVs,
550 Mass Spectrometry Reviews DOI 10.1002/mas
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PROTEOMICS IN BACTERIAL OUTER MEMBRANE VESICLES
which are made artificially from bacterial cell membranes (de
EM electron microscope
Moraes et al., 1992; Drabick et al., 1999; Girard et al., 2006).
ESI electrospray ionization
However, based on previous proteomic reports, the ratio of outer
FFE free-flow electrophoresis
membrane proteins present in DOMVs was relatively low
ICAT isotope-coded affinity tags
compared to native OMVs and was not consistent from batch
iTRAQ isobaric tags for relative and absolute
to batch, resulting in different and diverse immune responses in
quantification
the host depending on the manufacturer (Ferrari et al., 2006).
LC liquid chromatography
Therefore, vaccination strategies should also be envisioned
LPS lipopolysaccharide
using native OMVs, which carry the enriched and native
LT heat-labile enterotoxins
topology of strain-specific outer membrane antigens. Creating
MALDI-TOF matrix-assisted laser desorption/ionization
super-blebbing bacterial mutants that can produce several times
time-of-flight
more vesicles than wild-type strains and genetic mutants that can
MS mass spectrometry
control LPS amounts might reduce the side effects of septic
symptoms in patients and will provide a particularly useful MudPIT multidimensional protein identification
source for vaccine materials (van der Ley et al., 2001; Berlanda technology
Scorza et al., 2007). MV membrane vesicle
Another important application of OMVs comes in the area of
OMV outer membrane vesicle
antibiotics. Adverse reactions in patients undergoing therapy,
SILAC stable-isotope labeling by amino acids in cell
resulting from antibiotic-induced liberation of bacterial compo-
culture
nents, including OMVs, have been a long-standing concern
SDS PAGE sodium dodecyl sulfate polyacrylamide gel
(Kadurugamuwa & Beveridge, 1995; Morand & Muhlemann,
electrophoresis
2007). Moreover, treatment with broad-spectrum antibiotics can
disturb natural and beneficial microflora, leaving the patient more
susceptible to infection by opportunistic pathogens. Therefore,
ACKNOWLEDGMENTS
modulating the production of OMVs presents an attractive
approach for treating bacteria-associated diseases.
This work was supported by a grant of the National R&D
Program for Cancer Control, Ministry of Health & Welfare,
Republic of Korea (0320380-2), supported by the Korea Basic
Science Institute K-MeP (T27021), and supported by the Korea
VII. CONCLUDING REMARKS
Science and Engineering Foundation (KOSEF) grant funded by
the Korea government (MOST, No. R15-2004-033-05001-0) to
Growing evidence suggests that Gram-negative bacterial OMVs
Yong Song Gho. Eun-Young Lee and Dong-Sic Choi were
are essential for bacterial survival and pathogenesis in hosts by
acting as intercellular communicasomes in polyspecies com- recipients of Brain Korea 21 fellowship.
munities. In spite of recent progress in this emerging field,
previous biochemical and biological studies are limited in their
ability to provide comprehensive information for understanding
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Eun-Young Lee received a B.Sc. degree in the Division of Life Science from Korea
University, Republic of Korea (2005). Presently she is a Ph.D. candidate at Pohang
University of Science and Technology, Republic of Korea, with a focus on the diverse roles
of bacterial outer membrane vesicles and mammalian cell-derived microvesicles under the
supervision of Prof. Yong Song Gho.
Dong-Sic Choi received a B.Sc. degree in the Department of Life Science and Division of
Molecular and Life Sciences from Pohang University of Science and Technology, Republic
of Korea (2006). He is now a Ph.D. candidate at Pohang University of Science and
Technology and his research involves proteomic analysis of microvesicles and plasma
membrane proteins derived from eukaryotic cells under the supervision of Prof. Yong Song
Gho.
Kwang-Pyo Kim received his B.Sc. and M.Sc. in Chemistry from Seoul National
University, Republic of Korea in 1990 and 1992, respectively. After working for a
pharmaceutical company, CJ corp., he started a Ph.D. program in Chemistry from the
University of Illinois at Chicago. In 2002 he thereafter worked in Harvard Medical School
as a postdoctoral fellow. Since 2004, he joined the Department of Molecular Biotechnology
at the Konkuk University in the Republic of Korea as an Assistant Professor. His current
research areas focus on: development and application of proteomics technologies to
investigate post-translational modifications, discovery of biomarkers with comparative
proteomics technologies, and MALDI Mass Tissue imaging.
Yong Song Gho received his B.Sc. and M.Sc. degrees in Chemistry from Seoul National
University, Republic of Korea in 1987 and 1989, respectively. He obtained his Ph.D. degree
in Biochemistry and Biophysics from University of North Carolina at Chapel Hill, USA in
1997. During 1998 2000, he was a visiting fellow at NIDCR of National Institutes of
Health, USA. From 2000 to 2004, he was an assistant professor at Kyung Hee University,
Republic of Korea. Since 2004, he became an assistant professor in the Department of Life
Science and Division of Molecular and Life Sciences at Pohang University of Science and
Technology, Republic of Korea. His current research interests aim at elucidating the
biogenesis and pathophysiological functions of extracellular membrane vesicles derived
from bacteria and mammalian cells, as well as determining profiles of membrane vesicular
genes and proteins using microarray and mass spectrometry.
Mass Spectrometry Reviews DOI 10.1002/mas 555